Application of GFP Imaging in Cancer Robert M Hoffman

PATHOBIOLOGY IN FOCUS

Application of GFP imaging in cancer Robert M Hoffman

Multicolored proteins have allowed the color-coding of cancer cells growing in vivo and enabled the distinction of host from tumor with single-cell resolution. Non-invasive imaging with fluorescent proteins enabled the dynamics of metastatic cancer to be followed in real time in individual animals. Non-invasive imaging of cancer cells expressing fluorescent proteins has allowed the real-time determination of efficacy of candidate antitumor and antimetastatic agents in mouse models. The use of fluorescent proteins to differentially label cancer cells in the nucleus and cytoplasm can visualize the nuclear–cytoplasmic dynamics of cancer cells in vivo including: mitosis, apoptosis, cell-cycle position, and differential behavior of nucleus and cytoplasm that occurs during cancer-cell deformation and extravasation. Recent applications of the technology described here include linking fluorescent proteins with cell-cycle-specific proteins such that the cells change color from red to green as they transit from G1 to S phases. With the macro- and micro-imaging technologies described here, essentially any in vivo process can be imaged, giving rise to the new field of in vivo cell biology using fluorescent proteins. Laboratory Investigation (2015) 95, 432–452; doi:10.1038/labinvest.2014.154; published online 16 February 2015

‘...during periods of revolution when the fundamental tenets cancer cells of a specific genotype or phenotype. For example, of a field are once more at issue, doubts are repeatedly the behavior of cancer stem cells labeled with GFP and non- expressed about the possibility of continued progress if one stem cells labeled with red fluorescent protein (RFP) can be or another of the opposed paradigms is adopted.’ simultaneously compared in vivo.3 The stroma and the cancer Thomas S. Kuhn in The Structure of Scientific Revolutions cells can be color-coded with fluorescent proteins. For (3rd Edition, The University of Chicago Press, Chicago and example, a transgenic mouse expressing GFP in all of its London, 1996). cells (or in specific cells such as endothelial cells) transplanted with cancer cells expressing RFP enables the inter- INTRODUCTION action between the cancer cells and the host cells to be 4–6 When the Nobel Prize in chemistry was awarded for the visualized in real time. discovery of green fluorescent protein (GFP) in 2008, the The long wavelength excitation of some fluorescent pro- 7 Nobel Committee called GFP a guiding star for biochemistry teins, such as Katushka, also reduces the extent of photo- enabling processes that were previously invisible, such as bleaching and enables deep imaging. Therefore, real-time cancer cells spreading, to be strikingly visible. GFP and its red, imaging of tumor growth and metastasis can be carried out blue, yellow, and orange cousins have revolutionized bio- in the intact animal in deep tissues. For single-cell resolution, medical research and enabled every major disease, both cause reversible skin flaps as well as chronic-transparent window and effect, to ‘light-up’ in the laboratory for researchers to models can be used over many parts of the body (skin, brain, 8–10 visualize, understand, and eventually conquer them. Fluor- lung, liver, etc.). Real-time imaging with fluorescent proteins escent proteins allow us to visualize, in real time, all important is especially important when evaluating the efficacy of thera- 11 aspects of cancer in living animals, including the cancer cell cycle, peutics on metastasis, tumor recurrence, and cell cycle 12 mitosis, apoptosis, motility, invasion, metastasis, and angio- dependence. genesis. Multicolored proteins have allowed the color-coding of cancer cells growing in vivo and enabled the distinction of Properties of GFP and Other Fluorescent Proteins as host from tumor with single-cell resolution in vivo.1,2 Imaging Agents Fluorescent proteins of many different colors have now GFP and related fluorescent proteins are a family of proteins been characterized and these can be used to color-code with emission spectra ranging from 442 to over 650 nm.13

AntiCancer, Inc., Department of Surgery, University of California San Diego, San Diego, CA, USA Correspondence: RM Hoffman, PhD, AntiCancer, Inc., Department of Surgery, University of California San Diego, 7917 Ostrow Street, San Diego, CA 92111, USA. E-mail: [email protected] Received 14 August 2014; accepted 25 September 2014

432 Laboratory Investigation | Volume 95 April 2015 | www.laboratoryinvestigation.org PATHOBIOLOGY IN FOCUS GFP imaging and cancer RM Hoffman

There are many spectrally distinct fluorescent proteins are being used. Spectral resolution enables imaging of tumor resulting in a palette of colors14 that can be simultaneously on deep tissues or high-resolution non-invasive visualization imaged. Fluorescent proteins range in size from 25 to 30 kDa of tumor blood vessels16 which express a fluorescent protein and form internal chromophores that do not require cofactors of a different color. or substrates to fluoresce. Fluorescent proteins generally have very high extinction coefficients ranging up to approximately Ex Vivo Imaging Using Fluorescent Proteins ¼ 95 000. In addition, fluorescent proteins have very high The first use of GFP for in vivo imaging, visualized cancer 15 quantum yields up to 0.8. These properties make cells growing in athymic nude mice in our laboratory.21 fluorescent proteins very bright. Two-photon absorption of Cancer cells were stably transfected with GFP and were trans- 10 GFP is important for deep-tissue imaging in vivo. Another planted into several mouse models, including orthotopic important feature of fluorescent proteins is the spectral models that have a high metastatic capacity. Metastases distinction between many members of the family, enabling of could be observed in any organ at the single-cell level in multicolor fluorescent proteins to be used simultaneously for unprocessed tissues. In addition, cells were visualized in the multifunctional in vivo imaging. Spectral separation imaging process of intravasation and extravasation. The visualization is also very useful to distinguish different colors including of single cancer cells in tissue is beyond the capabilities of 16 autofluorescence. standard histological techniques.

In Vivo Imaging with Fluorescent Proteins Fluorescent proteins are so bright that simple equipment can Intravital Imaging Using Fluorescent Proteins be used for in vivo imaging. Macro-imaging studies require Before our laboratory’s pioneering use of GFP for in vivo equipment as simple as an LED flashlight with appropriate imaging, in vivo fluorescence imaging was limited to the vi- excitation emission filters.17 In vivo images can even be sualization of cells that were transiently labeled with vital acquired with a cell phone camera because fluorescent dyes. Use of fluorescent proteins, now allows the direct proteins are so bright! imaging of single cells in vivo for unlimited periods. A fluorescence light box with fiber-optic lighting at ap- Reversible skin flaps can be introduced onto different parts proximately 490 nm and appropriate filters, placed on top of of the animal to image single cancer cells or small colonies on 8 the light box (Figure 1), can be used to image tumors and internal organs. The skin flaps are reversed by simple metastasis that can be viewed with a camera to enable the suturing. External imaging can then be done through the images to be displayed on a monitor and digitally stored.18 relatively transparent body walls of the mouse, which include Excitation with a narrow band filter at approxi- the skull, at the single-cell level, by use of a simple mately 490 nm should be used. Fluorescence emission can fluorescence dissecting microscope or more sophisticated be observed through a 520 nm long-pass filter.18 equipment described in this review. Blood vessels growing on A powerful hand-held imaging device that inputs the image tumors can also be observed using skin-flaps because they 8 directly to a computer monitor can also be used.19 contrast with the fluorescence of the tumors. 22 A variable-magnification small animals imaging system With intravital microscopy, imaging through the opened (OV100, Olympus Corp., Tokyo, Japan), containing an MT- skin or body walls of live mice visualizes single cancer cells. All 20 light source (Olympus Biosystems, Planegg, Germany) steps in the metastatic process can be visualized. Individual, and DP70 CCD camera (Olympus), can be used for macro- nondividing cells as well as micro- and macrometastases can and subcellular imaging in live mice. The optics of the OV100 be clearly visualized and quantified. Cellular details, such as 23 fluorescence imaging system have been specially developed pseudopodial projections, can be clearly seen. In early 24 for macroimaging as well as microimaging with high light- studies, Farina et al observed cancer cell invasion at the gathering capacity. The objectives have high numerical single-cell level, including intravasation and extravasation, 25 aperture and long working distance. Individually optimized using GFP-expressing cells. Condeelis et al used confocal objective lenses, parcentered and parfocal, provide a 105-fold GFP imaging to observe the polarity of cancer cells magnification range for imaging of the entire body down to responding to chemotatic cytokines by intravital imaging. the subcellular level without disturbing the animal. The OV100 has lenses mounted on an automated turret with a Multiphoton Laser-Scanning Microscopy high magnification range of Â 1.6 to Â 16 and a field of view Multiphoton laser-scanning microscopy26 can provide ranging from 6.9 to 0.69 mm. The optics and anti- high-resolution to image deeper regions of GFP-expressing reflective coatings ensure optimal imaging of multiplexed tumors.23,24 Fukumura et al27,28 monitored the activity of the fluorescent reporters in small animals (Figure 1).20 vascular endothelial growth factor promoter in transgenic The use of tunable filters allows the isolation of any in- mice that expressed GFP under the control of this promoter. dividual spectrum in any fluorescent pixel. This technique Multiphoton laser-scanning microscopy showed that the eliminates autofluorescence as well as enabling high-resolu- tumor was able to induce activity of this promoter and tion spectral distinction when multiple fluorescent proteins subsequent blood-vessel formation.27,28

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Figure 1 Imaging apparatus from simple to complex. (a) An example of the earliest prototype for in vivo imaging with GFP is the Illumatool, a simple instrument with light sources that are properly filtered to avoid autofluorescence and an emission filter through which it is possible to image GFP fluorescence from unrestrained animals.18 (b) Whole-body imaging of GFP- and RFP-expressing tumors implanted in the brain in a single nude mouse. The excitation light was produced with a simple blue-LED flashlight equipped with an excitation filter with a central peak of 470 nm (shown in the figure). The image was acquired with a Hamamatsu charge-coupled device (CCD) camera. (c) An example of the best state of the art, OV100 Small Animal Imaging System (Olympus, Tokyo, Japan). The OV100 contains an MT-20 light source (Olympus) and DP70 CCD camera (Olympus). The optics of the OV-100 fluorescence imaging system have been specially developed for macroimaging as well as microimaging with high light-gathering capacity. The instrument incorporates a unique combination of high numerical aperture and long working distance. Individually optimized objective lenses, parcentered and parfocal, provide a 105-fold magnification range for seamless imaging of the entire body down to the subcellular level without disturbing the animal. The OV-100 has the lenses mounted on an automated turret with a high magnification range of Â1.6 to Â16 and a field of view ranging from 6.9 to 0.69 mm. The optics and antireflective coatings ensure optimal imaging of multiplexed fluorescent reporters in small animals. High-resolution images are captured directly on a PC (Fujitsu Siemens, Munich, Germany).20

Color-Coded Imaging of Tumor Angiogenesis endothelial cell precursors. For state-of-the-art information Following transplantation of the RFP-expressing murine on nestin-GFP expression, please see Matsuzaki et al.30 melanoma cell line B16F10 into transgenic mice in which the promoter of the gene coding for the stem cell-marker nestin Imaging GFP-Labeled Cancer Cells in Blood Vessels drives GFP (ND-GFP), angiogenesis was visualized using Cancer cell trafficking in blood vessels was originally observed in dual-color fluorescence imaging. This was possible because real time after injection of GFP-expressing cancer cells in the ND-GFP was expressed in the proliferating endothelial cells tail vein of mice.31 Lung carcinoma cells32 and mammary and nascent blood vessels in the growing tumor which was cancer cells,33 both expressing GFP, interacted with the blood- labeled with RFP. Color-coded imaging demonstrated that vessel wall as seen via skin window chambers in rodents. doxorubicin inhibited tumor angiogenesis as well as tumor Angiogenesis was induced by as few as 60–80 cancer cells. In growth in these mice.29 The ND-GFP stem cells are the surrounding tissue, increased vasodilation and changes

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in blood-vessel morphology were also observed. Angiogenesis behavior in blood vessels. The metastatic cells had a greater was also visualized using a GFP-expressing gliosarcoma affinity for blood vessels. In contrast, the non-metastatic rodent cell line.34 cells lost part of their cytoplasm when interacting with the Hematogenous metastases containing GFP cancer cells vessel wall, possibly as a result of the shearing of cell pseu- were visualized in intact perfused mouse and rat lung. The dopods that were inserted into the blood flow during GFP-expressing cells were visualized attached to the endo- intravasation.25 thelia of pulmonary precapillary arterioles and capillaries as 35,36 well as in colonies formed within the blood vessels. Cancer Cell Trafficking in Lymphatic Channels Imaged PC-3-GFP human prostate cancer cells metastasized from with RFP and RFP the orthotopic site to within the blood vessels of the liver and GFP/RFP-labeled cancer cells trafficked through lymphatic the lung, where they proliferated. The intravascular cancer vessels where they were imaged via a skin flap in real time at cells produced their own microenvironment, where they the cellular level until they entered an axillary lymph node.41 could continue to proliferate. Extravasation occurred earlier 37 in the lung than in the liver. Transdifferentiation of Cancer Cells into Blood Vessel In order to study cytoplasmic and nuclear deformation, Cells dual-color cancer cells expressing GFP in the nucleus and GFP-labeled colon carcinoma were also visualized interacting RFP in the cytoplams were injected intracardially. Cancer with blood vessels. These tumors had mosaic vessels with cells which reached capillaries elongated to fit the width of focal regions in which no CD31- or CD105-expressing these vessels. The average length of the major axis of the immunoreactivity was detected and cancer cells appeared to cancer cells in the capillaries increased to 3.97 times their contact the vessel lumen. The cancer cells appeared to form normal length and nuclei increased their length 1.64 times in part of their own vasculature.42 Transplantation of glioma the capillaries. Non-dividing cancer cells in capillaries over 38 cells expressing GFP in transgenic mice ubiquitously express- 8 mm in diameter could migrate up to 48.3 mm/h. The limits ing RFP demonstrated that tumor-derived endothelial cells of cell migration may depend on deformation of the nucleus, originated from tumor-initiating cells and did not result as shown with the dual-color cancer cells expressing GFP in 39 from cell fusion of endothelial cells and cancer cells. Thus, the nucleus and RFP in the cytoplasm. cancer cells differentiated to endothelial cells which formed The nuclear and cytoplasmic behavior of dual-color cancer blood vessels within the tumor.43 cells with GFP in the nucleus and RFP in the cytoplasm were imaged in real time in large blood vessels as they moved Cancer Neurogenesis by various means or adhered to the vessel surface in the In ND-GFP mice, GFP is also expressed in neural stem cells abdominal skin flap. During extravasation, real-time dual- as well as nascent blood vessels. A human intramedullary color imaging showed that cytoplasmic processes of the spinal cord tumor transplanted to ND-GFP nude mice at- cancer cells exited the vessels first, with nuclei following tracted nestin-GFP cells expressing neuronal class III-b-tu- along the cytoplasmic projections. Both cytoplasm and nuclei bulin as well as CD31 expressing cells. Both cell types underwent deformation during extravasation.20 surrounded the tumor. These results suggest that the tumor HT-1080 human fibrosarcoma cells, were labeled with RFP stimulated both neurogenesis as well as angiogenesis.44 (mCherry), linked to histone H2B, was expressed in the nucleus and GFP expressed in the cytoplasm. Cancer cells were injected in the epigastric cranialis vein in an abdominal Imaging Metastatic Cells with GFP flap of nude mice as an imaging model. The cell cycle posi- The initial method used to introduce cancer cells into tion of individual living cells was visualized by the nuclear– an animal model can affect the metastatic processes, with cytoplasmic ratio and nuclear morphology. Real-time in- surgical orthotopic implantation (SOI) of tumor fragments 45 duction of apoptosis was observed by nuclear size changes resulting in the most patient-like model. and progressive nuclear fragmentation. Intra- and extravascular mitotic cells were also visualized. One hour after cell Imaging Bone Metastasis injection, round and elongated cancer cells were observed in A GFP-expressing MDA-MB-435 human breast carcinoma the vessels. Five hours after injection, dual-color cancer cells cell line produced widespread osteolytic skeletal metastases began to divide within the vessel. By 10 h, some intravascular following injection into the left ventricle of the heart.46 GFP cancer cells underwent apoptosis. Deformed new blood ves- expression also permitted ex vivo detection of single cells and sels in the tumor were observed 10 days later. Extravascular microscopic metastases in bone at early time points. cancer cells were imaged dividing in the tumor at day 14 after Fragments of subcutaneously growing H460-GFP lung injection.40 tumors were implanted by surgical orthotopic implantation GFP-expressing metastatic (MTLn3) and non-metastatic in the left lung of nude mice. Subsequent micrometastases (MTC) cell lines, derived from the rat mammary adeno- were visualized by GFP fluorescence in the contralateral lung, carcinoma 13762 NF cell line, were compared for their plural membrane and widely throughout the skeletal system

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including the skull, vertebra, femur, tibia, pelvis, and bone bone were inhibited. Using whole-body GFP imaging, Peyru- marrow of the femur and tibia.47 chaud et al54 also showed that the angiogenesis inhibitor B16 GFP cells were i.v. injected in C57BL/6 mice and angiostatin inhibited tumor growth in bone by inhibiting human LOX GFP melanoma cells intradermally injected in osteoclast activity. nude mice. Extensive bone and bone marrow metastases of B16 were visualized by GFP expression when the animals Brain Metastasis were killed. Metastases of both cell lines were visualized in In early experiments, the human lung adenocarcinoma many organs, including the brain, lung, pleural membrane, cell-line Anip 973 was transfected with GFP and stable liver, kidney, adrenal gland, lymph nodes, skeleton, muscle, high-level GFP-expressing transfectants were established. and skin by GFP fluorescence.48 GFP-expressing cells were injected intravenously in nude PC-3-GFP prostate cancer cells produced extensive bone mice as an experimental metastasis model. Intravenously lesions when injected into the tibia of immunocompromised injected mice had metastases in the brain and other organs as mice. The skeletal progression of the PC-3 cells could be detected by GFP expression in fresh tissue.55 monitored by GFP imaging, X-ray, and by measurements of In subsequent experiments, bright and highly stable GFP- tumor products in serum, including parathyroid hormone- expressing transfectants of the B16 mouse malignant-melano- related protein (PTHrP) and osteoprotegrin. The bispho- ma cell line and the LOX human-melanoma line were made. sphonate, pamidronate, reduced tumor burden as assessed by Metastases for both cell lines were visualized in many organs as imaging and biomarkers.49 described above, including the brain by GFP fluorescence.48 In a subsequent study, another bisphosphonate, 3,3-di- PC-3-GFP growing orthotopically in nude mice formed methylaminopropane-1-hydroxy-1,1-diphosphonic acid (ol- micrometastases and metastases in the central nervous system padronate) was the most effective bisphosphonate in in many organs, including the brain and spinal cord as vi- reducing tumor burden as assessed by GFP imaging and sualized by GFP imaging.56 radiography. The GFP tumor area and X-ray score sig- Androgen-dependent variants of the LNCaP human nificantly correlated. Reduced tumor growth in the bone was prostate cancer were developed, which were more tumori- accompanied by reduced serum calcium, PTHrP, and os- genic and exhibited invasion and metastasis compared with teoprotegrin.50 the parent LNCaP cells. Orthotopic transplantation of the PC-3-GFP cells were transfected to express different forms androgen-independent variants of LNCaP expressing GFP of PTHrP and were injected intraskeletally. Skeletal progres- resulted in multi-organ metastasis including the brain.57 sion of the prostate cancer cells was evaluated radiologically Orthotopic transplantation of the murine Lewis lung and by measurement of serum tumor markers. PTHrP carcinoma (LLC)-GFP resulted in rapid tumor growth in transfection converted a non-invasive cell line into one that nude mice and extensive metastasis visualized by GFP progressed in the skeleton: Injection of the PTHrP-trans- expression. Brain metastases were visualized in 30% of the fected cells resulted in greater tumor progression in bone animals.58 when compared with non-transfected cells, and this effect LLC cells, stably expressing GFP, were injected in the chick was also influenced by non-amino terminal peptides of embryo chorioallantoic membrane. GFP-LLC metastases PTHrP.51 were visualized by fluorescence, 7 days additional incubation, Increased tumor progression was observed in PTHrP- in the brain and other organs, with the most frequent site overexpressing DU 145-GFP cells, whereas decreased pro- being the brain.59 gression was observed by PTHrP-knockdown in PC-3-GFP An orthotopic retinoblastoma (RB) model was established cells. PTHrP-overexpressing DU 145-GFP formed large tumors with human RB cells expressing GFP. RB-GFP cells were injected when implanted orthotopically into nude mice and resulted into the subretinal space of nude mice. The growth of trans- in spinal metastasis in one mouse, which was not observed in planted RB was observed in vivo using fluorescence stereo- mice implanted with parental DU 145 cells. PTHrP-over- microscopy. Metastasis to the cranium along the optic nerve expressing DU 145-GFP cells also caused significant bone was observed, with green fluorescent RB cells migrating along destruction when injected into the tibiae of nude mice, unlike the optic nerve sheath and long posterior ciliary artery.60 parental DU 145 cells.52 Two nasopharyngeal carcinoma cell lines stably expressing Peyruchaud and colleagues53 established a GFP-expressing GFP, 5-8F-GFP and 6-10B-GFP, were established. The cells bone-metastasis subclone of MDA-MB-231 breast cancer were orthotopically injected into the nasopharynx. Whole- cells (B02/GFP.2) that grows preferentially in bone. Whole- body fluorescence imaging was used to monitor the growth body fluorescence imaging of live mice showed that bone of the primary tumor as well as angiogenesis and metastasis, metastases could be detected about a week before radio- which demonstrated brain metastasis of 5-8-F GFP. Cell line logically distinctive osteolytic lesions developed. Further- 6-10B was less metastatic, but occasionally resulted in pul- more, when the tumor-bearing mice were treated with a monary metastasis. GFP enabled imaging of micrometastasis. bisphosphonate, the progression of established osteolytic 5-8F was highly sensitive to 5-fluorouracil, whereas 6-10B lesions and the expansion of the breast cancer cells in the was moderately sensitive.61

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To develop an imageable, patient-like model of spinal cord glioma, U87 human glioma tumor fragments expressing RFP grown in nude mice were transplanted by surgical orthotopic implantation into the spinal cord of non-transgenic nude mice or transgenic nude mice expressing ND-GFP as described above. The intramedullary spinal cord tumor grew at the primary site, caused hind-limb paralysis, and also metastasized to the brain.44 Primary tumor size was significantly reduced by temozolomide treatment compared with untreated controls and brain metastases were found only in the control group. Histological analysis of the control group showed aggressive tumor invasion in the spinal cord. In contrast, temozolomide-treated animals showed mostly scar tissue after tumor transplantation.44 Multiphoton laser scanning microscopy was used to image brain metastasis formation in real time at the single-cell level. Brain metastases arrested at vascular branch points and were closely associated with microvessels. Long-term dormancy was observed for some of the brain metastatic cells.62 Estrogen-receptor-positive breast cancer cells expressing GFP, resistant to puromycin and non-tumorigenic without exogenous estrogen, were injected intracardially into mice that already had growing orthotopic tumors formed by estrogen-receptor -negative cells resistant to the antibiotic G418. A variant cell line containing both estrogen-dependent and -independent cells was isolated from GFP-expressing cells in the bone marrow, which was resistant to both puromycin and G418, suggesting its origin from the fusion of the two cells types. The fused cells were also metastatic to the brain detected by GFP, unlike parental cells. It was suggested that cell fusion can contribute to brain metastasis ability.63 The effect of UVC irradiation was investigated on a model of experimental brain metastasis. For the brain metastasis model, lung cancer cells were injected intra-carotidally or stereotactically. The LLC line was used for the experimental brain metastasis model. The cancer cells were double-labeled with GFP in the nucleus and RFP in the cytoplasm. A craniotomy open window was used to image single cancer cells in the brain. The double-labeling of cancer cells with GFP and RFP enabled apoptosis of single cells to be imaged at the subcellular level through the craniotomy open window in live mice in real time. UVC irradiation, beamed through the craniotomy open window, induced apoptosis in the cancer cells. UVC irradiation was effective on LLC and significantly extended survival of the mice with experimental brain metastasis (Figure 2).64 The dual-color LLC cells were imaged through the cra- Figure 2 Real-time imaging of apoptotic cell death in the brain in live niotomy open window 10 days after treatment with temo- mice. (a) Seven days after dual-color Lewis lung carcinoma (LLC) cell zolomide. After treatment, dual-color cancer cells with inoculation via the internal carotid artery, the cells were irradiated for fragmented nuclei were visualized, indicating apoptosis. 60 s with UVC. (b) Three hours after irradiation, aggregation of chromatin GFP-expressing apoptotic bodies and the destruction of RFP- at the nuclear membrane (arrow) and fragmented nuclei (arrowheads) expressing cytoplasm were also visualized in real time in nude were observed. (c) Six hours after irradiation, the nuclei appeared to form 65 numerous additional fragments and destruction of RFP-expressing mice through the craniotomy open window. cytoplasm was observed (scale bars, a,b: 100 mm; c: 20 mm).64 Mitosis of the individual metastatic cancer cells on the brain were imaged dynamically in real time through the

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Figure 3 Time-course dynamic imaging of a dual-color Lewis lung carcinoma (LLC) cell undergoing mitosis in the brain visualized through a craniotomy window with the OV100 imaging system. Arrows point to the same cell undergoing mitosis.66 craniotomy open window. This model can be used to eval- cells and the cancer cells were coinjected in the pv, liver uate brain metastasis and brain cancer at the subcellular level metastasis resulted that contained GFP spleen cells. These including their sensitivity to currently used and experimental results suggest that the splenocytes are not just passengers but drugs (Figure 3).66 are required for liver metastasis.71 BALB/c or SCID mice were injected intracardially with Subsequent studies have shown that the metastatic either 4T1-GFP or MDA231BR-GFP breast cancer cells, cells can bring their own stromal components, including and they subsequently formed brain metastasis. Antibody activated fibroblasts, from the primary site to the neutralization of either ALCAM or VLA-4 significantly lungs. Viability of circulating metastatic cancer cells is higher reduced tumor seeding within the brain, suggesting that if they are in emboli with stroma and have a growth ALCAM/ALCAM and VLA-4/VCAM-1 interactions are advantage in the lung, confirming that stromal cells drive important in the early stages of metastasis seeding in the metastasis.72 brain.67 Human HCT-116-GFP-RFP colon cancer and mouse The mTOR inhibitor, rapamycin and Temsirolimus- mammary tumor (MMT-RFP-GFP) cells were imaged in the CCI-779, inhibited brain metastasis of MDA-MB-231-BR pv of nude mice. Most HCT-116-GFP-RFP cells remained in and CN34-BrM2 cancer cells expressing GFP.68 Sunitinib and sinusoids near peripheral portal veins. Only a small fraction dasatinib were visualized to inhibit brain metastases derived of the cancer cells invaded the lobular area. Extensive clas- from human breast cancer MDA-MB-231-BR cells expressing mocytosis of the HCT-116-GFP-RFP cells occurred within GFP.69 4T1-BrM5-GFP breast cancer cells administered into 6 h. Apoptosis was imaged in the dual-color cells by their mammary fat pads of Balb/c mice subsequently formed brain altered nuclear morphology. In contrast, dual-color MMT- metastasis. The substance P inhibitor spantide III reduced GFP-RFP cells injected into the pv mostly survived in the cancer cell colonization in the brain. Substance P inhibition liver of nude mice 24 h after injection. The cells grew in combination with other therapies may prevent breaching aggressively and formed colonies in the liver. However, when the blood-brain barrier by breast cancer cells and their the host mice were pretreated with cyclophosphamide, the colonization in the brain.70 Sunitinib and dasatinib are HCT-116-GFP-RFP cells also survived and formed colonies multi-kinase inhibitors that may target metastasis.69 in the liver after pv injection. These results suggest that a cyclophosphamide-sensitive host cellular system attacked the Role of Stroma and Other Host Cells in Metastasis HCT-116-GFP-RFP cells but could not effectively kill the Human colon cancer cells that express GFP in the nucleus MMT-GFP-RFP cells. This cyclophosphamide-sensitive cel- and RFP in the cytoplasm (HCT-116-GFP-RFP) were lular system appears to inhibit metastasis of some cancer cell injected in either the portal vein (pv) or spleen of transgenic types and not others.73 nude mice expressing GFP and imaged at the subcellular level In another study that shows cyclophosphamide pre- in vivo. Extensive clasmocytosis (destruction of the cyto- treatment can drive metastasis, HT1080-GFP-RFP human plasm) of the cancer cells occurred within 6 h after pv injec- fibrosarcoma cells with GFP in the nucleus and RFP in the tion and essentially all the cancer cells died. In contrast, cytoplasm were injected into the epigastric cranialis vein of splenic injection of these cancer cells resulted in the nude mice. Twenty-four hours before cancer cell injec- aggressive formation of liver and distant metastasis. GFP tion, cyclophosphamide was given i.p. and greatly increased spleen cells were found in the liver metastases that resulted metastasis of HT-1080 GFP-RFP cells. Cyclophosphamide from intrasplenic injection of the cancer cells. When spleen seems to interfere with a host process that inhibits intra-

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vascular proliferation, extravasation, and extravascular colony The PC-3-GFP CTCs were then expanded in culture in formation of cancer cells, which are important events in meta- parallel with the parental PC-3-GFP cell line. Both cell types stasis. Cyclophosphamide does not directly affect the cancer were then inoculated onto the chorioallantoic membrane of cells because cyclophosphamide was cleared by the time the chick embryos. Eight days later, embryos were harvested and cancer cells were injected.74 the brains were processed for frozen sections. The IV100 intravital laser scanning microscope enabled rapid identifi- Identifying Clonality of Metastasis with GFP and RFP cation of fluorescent metastatic foci within the chick embryo- Imaging nic brain. Inoculation of embryos with PC-3-GFP CTCs GFP-labeled and RFP-labeled HT-1080 human fibrosarcoma resulted in a 3–10-fold increase in brain metastasis when cells were co-injected in the tail vein in order to determine compared with the parental PC-3-GFP cells.81 clonality by fluorescence visualization of metastatic colonies. The PC-3-GFP CTC had a significantly increased sensitivity Pure red or pure green colonies were scored as clonal, whereas to both cisplatin and docetaxel when compared with PC-3 GFP mixed red and green colonies were non-clonal. The clonality parental cells, with docetaxel having the greater efficacy.82 of the lung metastases was found to be dependent on the number of cells injected. Spontaneous metastasis were often Imaging Quiescent or Dormant Cancer Cells clonal and experimental metastasis were often non-clonal.9,75 Fluorescent protein probes were developed, which label individual G1 phase nuclei red and those in S/G2/M phases Imaging Circulating Tumor Cells (CTCs) with GFP green. This system is called a fluorescence ubiquitination- GFP-expressing PC-3 implanted orthotopically produced based cell cycle indicator (FUCCI).12 viable CTCs, but tumors of the same lineage did not when FUCCI 3D confocal imaging demonstrated that cancer s implanted subcutaneously. GFP-labeled circulating cells were cells in G0/G1 phase in Gelfoam histoculture migrated more more metastatic than the RFP-labeled parental cells when rapidly and further than cancer cells in S/G2/M phases. Cancer 76 they were co-implanted in the prostate. cells ceased migrating when they entered S/G2/M phases and Mixtures of RFP- and GFP-expressing PC-3 human pros- restarted migrating after cell division when the cells re-entered tate carcinoma cells were implanted in the nude mouse G0/G1. Migrating cancer cells also were resistant to cytotoxic prostate and metastasized. Yellow fluorescent PC-3 cells were chemotherapy, because they were preponderantly in G0/G1, observed and isolated from the circulation. The yellow where cytotoxic chemotherapy is not effective (Figure 4).83 fluorescent phenotype was heritable and stably maintained by FUCCI imaging showed that approximately 90% of cancer cancer cells for many generations in vitro and in vivo. In the cells in the center and 80% of total cells of an established animals implanted with the yellow-fluorescing cells, 100% tumor are in G0/G1 phase. Similarly, approximately 75% of developed aggressive lung metastases. In contrast, when the cancer cells far from (4100 mm) tumor blood vessels of an GFP- and RFP-expressing parental cells were separately established tumor are in G0/G1. Longitudinal real-time inoculated into the mouse prostate, none of the animals imaging demonstrated that cytotoxic agents killed only developed lung metastasis. All co-implanted animals had proliferating cancer cells at the surface and, in contrast, had almost exclusively yellow fluorescent cells in the blood and little effect on quiescent cancer cells, which are the vast bone marrow. These results are consistent with the idea that majority of an established tumor. Moreover, resistant quies- spontaneous genetic exchange occurring between cancer cells cent cancer cells restarted cycling after the cessation of che- in vivo contributes to genomic instability and creation of motherapy (Figure 5a,b).84 highly metastatic cells.77 FUCCI imaging demonstrated that OBP-301 adenovirus In another study, using color-coded imaging of high-me- decoyed quiescent cancer cells to advance from G0/G1 to S/ tastatic and low-metastatic osteosarcoma, it was observed G2/M phases by mobilizing cell-cycle-related proteins. OBP- that the high-metastatic phenotype could be transferred to 301 decoyed quiescent cancer stem-like cells in tumor spheres the low-metastatic cells. The K-ras and possibly other genes and also decoyed tumors in vivo into S/G2/M phases where were involved in conferring the high-metastatic phenotype.78 they became chemosensitive.85 Tumor-targeting Salmonella Metastatic human prostate carcinoma cells selected for typhimurium A1-R also decoyed quiescent cancer cells to survival in the circulation have increased resistance to anoi- cycle and become chemosensitive (Figure 5d).86 kis, which is apoptosis induced by cell detachment.79 Using the GFP-expressing PC-3 orthotopic model and Imageable Color-Coded Tumor-Host Models immunomagnetic beads coated with anti-epithelial cell Dual-color fluorescence imaging can be effected by using adhesion molecule and anti-prostate specific membrane RFP-expressing tumors growing in GFP-expressing trans- antigen, GFP-expressing CTCs were isolated within 15 min genic mice. Tumor–stroma interaction, especially tumor- and were readily visualized by GFP fluorescence. It was induced angiogenesis and tumor-infiltrating lymphocytes can possible to immediately place the immunomagnetic-bead- be readily imaged in the model. GFP-expressing dendritic captured GFP-expressing PC-3 CTCs in three-dimensional cells were observed contacting RFP-expressing cancer Gelfoams sponge-gel cell culture, where they proliferated.80 cells with their dendrites. GFP-expressing macrophages were

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6 s Figure 4 Invasive cancer cells are predominantly in G0/G1. FUCCI-expressing cancer cells (5 Â 10 ) were placed on Gelfoam (1 Â 1 cm) in RPMI 1640 medium. (a) High-magnification real-time images of invading cancer cells cultured on Gelfoams for 48 h. Arrows show the direction of invading cancer cells. (b) Histogram shows cell cycle phase of invading cancer cells.83

observed engulfing RFP-expressing cancer cells. GFP histochemical staining showed that CD31 was expressed in lymphocytes were seen surrounding cells of the RFP tumor, the ND-GFP-expressing nascent blood vessels.89 which eventually regressed (Figures 6 and 7).4 The tumor microenvironment (TME) has an important HT-1080-GFP-RFP human fibrosarcoma cells were visua- influence on tumor progression. Six different implantation lized surrounded by host-derived lymphocytes and macro- models were developed to image the TME using multiple phages, both expressing GFP. It was possible to observe host colors of fluorescent proteins: (i) RFP- or GFP-expressing GFP macrophages contacting, engulfing, and digesting dual- HCT-116 human colon cancer cells were implanted sub- color HT-1080-GFP-RFP cells in real time. The dual-color cutaneously in cyan fluorescent protein (CFP)-expressing cancer cells were readily visible after being engulfed in the nude mice. CFP stromal cells from the subcutaneous TME GFP macrophages. Other cancer cells were visualized being were visualized interacting with the RFP- or GFP-expressing killed by lymphocytes (Figure 8).87 tumors. (ii) RFP-expressing HCT-116 cells were transplanted The Olympus IV100 Laser Scanning Microscope, with ultra- into the spleen of CFP nude mice, which resulted in ex- narrow microscope objectives (‘stick objectives’), was used for perimental metastases formed in the liver. CFP stromal cells non-invasive three-color whole-body imaging of GFP-RFP from the liver TME were visualized interacting with the RFP- expressing cancer cells with GFP in the nucleus and RFP in the expressing tumor. (iii) RFP-expressing HCT-116 cancer cells cytoplasm interacting with the GFP-expressing stromal cells in were injected in the tail vein of CFP-expressing nude mice, their transgenic nude-mouse host footpad. In this model, drug forming experimental metastases in the lung. CFP stromal response of both cancer and stromal cells in the intact live cells from the lung were visualized interacting with the RFP- animal was also imaged in real time. Tumor–host interaction expressing tumor. (iv) In order to visualize two different and cellular dynamics were non-invasively imaged, including tumors in the TME, GFP- and RFP-expressing HCT-116 mitotic and apoptotic tumor cells, stromal cells interacting with cancer cells were co-implanted subcutaneously in CFP-ex- the cancer cells, tumor vasculature, and tumor blood flow.88 pressing nude mice. A three-color TME was formed sub- Human tumors grew extensively in nestin-driven cutaneously in the CFP mouse, and CFP stromal cells were (ND-GFP) transgenic nude mice. ND-GFP was shown to be visualized interacting with the RFP- and GFP-expressing tu- highly expressed in proliferating endothelial cells and nascent mors. (v) In order to have two different colors of stromal blood vessels in the growing tumors, visualized by dual-color cells, GFP-expressing HCT-116 cells were initially injected fluorescence imaging as described above. Results of immuno- subcutaneously in RFP-expressing nude mice. After 14 days,

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Figure 5 Cell-cycle phase distribution of cancer cells at the tumor surface and center. (a) FUCCI-expressing MKN45 human stomach cancer cells were implanted directly in the liver of nude mice and imaged at 35 days. (b) Histogram shows the cell-cycle distribution in the tumor at 35 days after implantation. (c) Representative 3D reconstruction images of a nascent tumor at 35 days after implantation. Data are means (each group n ¼ 5). Scale bars represent 100 mm.84 (d) S.typhimurium A1-R decoys the cell cycle transit of quiescent cancer cells in tumors in vivo. Representative images of a cross-section of FUCCI-expressing MKN45 tumor xenograft treated with S.typhimurium A1-R or untreated control.86

the tumor, which consisted of GFP cancer cells and RFP liver metastasis was imaged. GFP-expressing host cells were stromal cells derived from the RFP nude mouse, was har- recruited by the metastatic tumors as visualized by fluores- vested and transplanted into the CFP nude mouse. CFP cence imaging. A desmin-positive area increased around and stromal cells invaded the growing transplanted tumor con- within the liver metastasis over time, suggesting CAFs were taining GFP cancer cells and RFP stroma. (vi) MMT cells recruited by the liver metastasis, which appeared to have a expressing GFP in the nucleus and RFP in the cytoplasm were role in tumor progression of the metastasis.91 implanted in the spleen of a CFP nude mouse. Cancer cells Patient pancreatic tumors were passaged orthotopically were imaged in the liver 3 days after cell injection. The dual- in transgenic nude mice ubiquitously expressing RFP. color dividing MMT cells and CFP hepatocytes, as well as The primary patient tumors acquired RFP-expressing stroma. CFP non-parenchymal cells of the liver were imaged inter- The RFP-expressing stroma included CAFs and tumor-asso- acting with the two-color cancer cells. CFP-expressing ciated macrophages (TAMs). Further passage to transgenic host cancer-associated fibroblasts (CAFs) (Figure 9) were nude mice ubiquitously expressing GFP resulted in tumors predominantly observed in the TME models developed in the that acquired GFP stroma in addition to their RFP stroma, CFP nude mouse (Figure 10).90 including CAFs and TAMs as well as blood vessels. The RFP Non-colored HCT-116 human colon cancer cells were in- stroma persisted in the tumors growing in the GFP mice. jected in the spleen of GFP nude mice which led to the Further passage to transgenic nude mice ubiquitously ex- formation of experimental liver metastasis. The TME of the pressing CFP resulted in tumors acquiring CFP stroma in

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addition to persisting RFP and GFP stroma, including RFP- and GFP-expressing CAFs, TAMs and blood vessels.92 After unlabeled primary patient tumors acquired GFP- expressing stroma by passage in GFP mice, subsequent liver metastases and disseminated peritoneal metastases carried the stroma from the primary tumor, and recruited additional GFP-expressing stroma, resulting in the very bright fluorescence of the metastasis. The GFP-expressing stroma included CAFs and TAM in both the primary and metastatic tumors.93 Patient pancreatic tumors previously passaged orthotopically into transgenic nude mice ubiquitously expressing GFP and subsequently to nude mice ubiquitously expressing RFP, where they acquired very bright GFP and RFP stroma, respectively, were then orthotopically passaged to non-transgenic nude mice. It was possible to image the Figure 6 Visualization of angiogenesis in live tumor tissue 3 weeks after s.c. injection of B16F10-RFP melanoma cells in the transgenic GFP mouse. brightly fluorescent tumors non-invasively longitudinally Well developed, host-derived GFP-expressing blood vessels are visualized as they progressed in the non-transgenic nude mice in the RFP-expressing mouse melanoma.4 (Figure 11).94

Figure 7 Visualization of host macrophage–tumor cell interaction in fresh tumor tissue. Images show host macrophages expressing GFP interacting with human PC-3-RFP prostate cancer cells on day 35 after orthotopic implantation of PC-3-RFP cells in the transgenic GFP nude mouse. (a) Host GFP macrophage (arrowhead) contacting RFP cancer cell (arrow). (b) GFP macrophage (arrowhead) engulfing RFP cancer cell (arrow). (c) RFP cancer cell (arrow) engulfed by GFP macrophage (arrowhead). (d) RFP cancer cell (arrows) digested by GFP macrophage (arrowhead). (Scale bars, 20 mm.)4

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Figure 9 Interactions (arrows) of host stromal GFP-expressing fibroblast cell (small arrow) and Dunning RFP-expressing rodent prostate cancer cells (large arrows) in live tumor tissue. Scale bar, 20 mm.121

Figure 8 Visualization of host macrophage-cancer cell interaction in live mice. (a) Host GFP macrophages (arrow, broken arrow) were visualized Figure 10 Dual-color mouse mammary tumor (MMT) cells with GFP in contacting HT-1080-GFP-RFP cells. Macrophage processes can be seen the cytoplasm and RFP in the nucleus, growing in the liver of a CFP extending into the HT-1080-GFP-RFP cells (arrowhead). Another nude mouse after splenic injection. Dual-color MMT cells formed tumors macrophage (broken arrow) contained cytoplasmic fragments after in the liver of a CFP mouse 28 days after splenic injection. CFP, non- digesting an HT-1080-GFP-RFP cell. (b) A macrophage (arrow) engulfing parenchymal liver cells (yellow arrows), and dual-color MMT cancer cells the RFP-expressing cytoplasm of an HT-1080 cell shown in contact with (red arrows) were visualized simultaneously. The image was taken with additional HT-1080-GFP-RFP cells (broken arrows). Bar ¼ 20 mm.87 an FV1000 confocal microscope. (Bar ¼ 50 mm).90

Color-Coding Cancer Stem Cells and Non-Stem Cells human hepatocellular carcinoma cells were considered as The differential behavior of cancer stem-like and non-stem cancer stem-like cells, and CD133 À Huh-7 cells were cells was simultaneously distinguished in the same tumor considered as non-stem cancer cells. CD133 þ cells were la- by real-time color-coded imaging. CD133 þ Huh-7 beled with GFP and CD133 À cells were labeled with RFP.

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GFP-cancer stem-like cells were highly tumorigenic Use of Fluorescent Protein Imaging to Evaluate and metastatic as well as very resistant to chemotherapy Therapeutics in vivo compared with RFP- non-stem cancer cells.3 Human pancreatic cancer cell line MiaPaCa-2-RFP formed widespread metastases after orthotopic implants in mice. Gemcitabine greatly improved survival by inducing transient tumor regression over the first 3 weeks. However, at this time, non-invasive imaging visualized tumor growth and dis- semination which occurred despite continued treatment, suggesting the development of tumor resistance to gemcitabine. Drug efficacy was followed noninvasively in real time by imaging the RFP-expressing tumor and metastases, and was confirmed by fluorescence open imaging of autopsy specimens.95 Mice with orthotopic, MiaPaCa-2 RFP pancreatic cancer were subsequently treated with gemcitabine on a low metronomic dose schedule (METG) or maximum tolerated dose (MTDG) schedule with or without sunitinib. Treatment with METG suppressed metastasis at multiple sites, an effect enhanced by sunitinib.96

Clinical Application and Potential Clinical Application of GFP Imaging The telomerase-specific replication-selective adenovirus expressing GFP (OBP-401) was able to detect viable human CTCs in peripheral blood ex vivo. The fluorescence signal was amplified only in viable, infected CTC by viral replication.97 OBP-401-GFP labeled both epithelial and mesenchymal CTCs, providing a non-invasive alternative to tissue biopsy or surgical resection of primary tumors for companion diagnostics.98 Fong et al99 used a herpes simplex virus (NV1066) vector that expresses the gfp gene to label, in a mouse model, metastatic lung tumor foci of less than 1 mm located in the pleural cavity. The tumor foci became fluorescent because NV1066 selectively replicates in cancer cells and expresses GFP. The fluorescently labeled tumors were detected by using a thoracoscopic endoscope system (which can be inserted into the thoracic cavity) equipped with fluorescent filters. Thus, fluorescent proteins have the potential for tumor diagnosis.

Figure 11 (a) Whole-body non-invasive imaging of human pancreatic cancer orthotopic tumor in non-transgenic nude mice. Mice were non-invasively imaged at day 21 (upper panel), day 30 (middle panel), and day 74 (lower panel). The tumors in the non-transgenic nude mice are in the F4 passage after F1, in NOD/SCID mice after patient surgery; F2, in transgenic green fluorescent protein (GFP)-expressing nude mice; and F3 in transgenic red fluorescent protein (RFP)-expressing nude mice. The tumor acquired GFP and RFP stroma in the F2 and F3 passages, respectively. Images were obtained with the Olympus OV100 Small Animal Imaging System. (b) Image of human pancreatic cancer tumor tissue resected from the F4 passage with RFP and GFP stroma. Image was obtained with an FV1000 confocal laser microscope. Yellow arrows indicate RFP-expressing cancer-associated fibroblast cells (CAFs). Blue arrows indicate GFP-expressing CAFs.94

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Figure 12 Fluorescence-guided surgical removal of peritoneal disseminated HCT-116 tumors after GFP labeling with OBP-401 telomerase-dependent GFP-containing adenovirus. Non-colored HCT-116 human colon cancer cells were injected into the abdominal space of nude mice. Ten days later, 1 Â 108 PFU of OBP-401 were i.p. injected. (a) Disseminated nodules were efficiently labeled and noninvasively visualized by GFP expression 5 days after virus administration. (b) Under general anesthesia, laparotomy was performed to remove intra-abdominal disease under GFP-guided navigation. (c) Disseminated nodules visualized by GFP-guided navigation were removed. (Scale bars: a–c, 10 mm). (d) Frozen section of resected HCT-116 disseminated nodules with fluorescence detection. (Scale bar, 500 mm). (e) H&E section of HCT-116 disseminated nodules shown in d. The box outlines a region of d and e analyzed in f. (Scale bar, 500 mm). (f) Detail of the boxed region of d and e. (Scale bar, 50 mm).100

Kishimoto et al100 selectively and accurately labeled existing with UVC. The average residual tumor area after FGS was tumors in mice with GFP using the OBP-401 telomerase- significantly smaller than that after BLS only. The BLS- dependent GFP-containing adenovirus (Figure 12). The treated mice had significantly reduced survival compared labeled tumors could then be resected under fluorescence with FGS- and FGS-UVC-treated mice for both relapse-free guidance. Tumors that recurred after fluorescence-guided survival and overall survival. FGS-UVC-treated mice surgery maintained GFP expression.101 Because the recurrent had increased relapse-free survival and overall survival cancer cells stably express GFP, detection of cancer recurrence compared with FGS-only treated mice with relapse-free and metastasis is also possible with OBP- 401 GFP labeling. survival lasting at least 150 days, indicating the animals were Maintenance of label in recurrent tumors is not possible with cured.104 nongenetic probes.102 U87-RFP human glioma cells, expressing RFP, were New In Vivo GFP Imaging Technology injected stereotactically into the nude mouse brain through a In order to image the fate of cancer-cell-derived exosomes in craniotomy open window. Resulting gliomas were resected orthotopic nude mouse models of breast cancer, GFP was under fluorescence guidance or by bright light surgery (BLS). linked to CD63, which is a general marker of exosomes. Almost all cancer cells were removed using fluorescence- Orthotopic nude-mouse models of breast cancer engineered guided navigation without damage to the brain tissue. In with CD63-GFP secreted exosomes into the tumor micro- contrast, brain tumors were difficult to visualize under BLS environment. These results suggested that tumor-derived and many residual cancer cells remained in the brain after exosomes may contribute to forming a niche to promote bright-light surgery.103 tumor growth and metastasis.105 Human MiaPaCa-2-GFP pancreatic cancer cells were Human 143B osteosarcoma cells expressing av integrin- transplanted orthotopically in nude mice. BLS was performed GFP were generated by transfection with an av integrin-GFP on all tumor-bearing mice. The residual tumors were vector. Gelfoams (5.5 mm) was transplanted subcutaneously resected using a hand-held portable imaging system in mice in transgenic RFP nude mice. The implanted Gelfoams treated with FGS. The surgical resection bed was irradiated became highly vascularized with RFP vessels. 143B cells

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expressing av integrin-GFP were injected into the vascular- proteins such as Katushka greatly reduce problems with ized Gelfoams. Cancer cells and blood vessels were imaged excitation, even in deep tissues, as shown by Shcherbo et al.7 in the vascularized Gelfoams by color-coded confocal In comparison with the luciferase reporter, GFP has a much microscopy via a skin flap. Strong expression of av integrin- stronger signal and can therefore be used to image unre- GFP in 143B cells was observed near RFP vessels in the strained animals down to the subcellular level with non- Gelfoams.106 damaging blue light. The RFP signal was approximately 1000 When the 143B av integrin-GFP cells were orthotopically times stronger than that of the luciferase reporter.111 Real- transplanted into the tibia of nude mice, the cells aligned time images from fluorescent proteins in vivo can be captured along the collagen fibers within the tumor and had punctuate using fairly simple apparatus and there is no need for total expression of av integrin-GFP. Invading osteosarcoma cells darkness.113 had punctate av integrin-GFP in the muscle tissue at the In vivo GFP images can be captured using even an LED primary tumor margin.107 flashlight with a proper excitation filter and an emission filter Using color-coded imaging, the interaction between GFP- with costs as low as a few hundred dollars17 in comparison expressing mesenchymal stromal cells and RFP-expressing with hundreds of thousands of dollars worth of equipment 4T1 mouse mammary tumor cells was monitored. As few as necessary for luciferase imaging. five 4T1 cells could give rise to tumor formation when A novel fusion protein has been developed, ffLuc-cp156, co-injected along with mesenchymal stromal cells into the which consists of a yellow variant of Aequorea GFP and mouse mammary fat pad, but no tumor was formed when firefly luciferase that has a three orders of magnitude increase 5 or 10 4T1 cells were implanted alone. Mesenchymal stromal in photon yields over current luciferase systems and should cells created a vascularized environment that enhances the prove to be very useful for in vivo imaging by allowing the ability of 4T1 cells to colonize and proliferate.108 advantages of both types of imaging simultaneously.115 After tail vein injection of dual-color HT1080 RFP-GFP Be careful of misconceptions in the literature. For example, cancer cells, they could be imaged seeding and growing on the following was recently published ‘Whole animal fluor- the lung by open-chest assisted-ventilation imaging. The escence imaging in vivo suffers from low signal-to-noise as a seeding and arrest of single cancer cells on the lung, accu- result of background autofluorescence, modeling-dependent mulation of cancer-cell emboli, cancer-cell viability, and photon quantification, photobleaching, low tissue penetra- metastatic colony formation were imaged in real time by tion, and low resolution’.116 Despite enormous evidence open-chest imaging.109 to the contrary, as outlined above, this type of misleading Dual-color imaging has also visualized RFP- and GFP- misinformation continues to be published even in 2013. expressing blood vessels anastomosing (Figure 15).110 GFP, RFP, and CFP transgenic nude mice appear to have a life span similar to that of non-transgenic nude mice, such CONCLUSIONS that long-term tumor growth and metastasis studies can be Fluorescent Proteins are the Method of Choice for carried out.5,117 Thus, fluorescent proteins are not toxic to Whole-Body Imaging the animal (Figure 13). The features of fluorescent-protein-based imaging, such as a Non-invasive subcellular imaging88 is a significant impro- very strong and stable signal, enable noninvasive whole-body vement from the inserted-window models22 or abdominal imaging down to the subcellular level,88 especially with red- imaging windows118 or of the skin flap models we have shifted proteins, and make it far superior to luciferase-based previously developed to image tumors.4,20 imaging. Luciferase, with its very weak signal,111 precluding Cell and tissue culture can be performed on different image acquisition and allowing only photon counting with substrates such as on plastic, in Matrigelt, and on Gelfoams, pseudocolor-generated images, has very limited applica- a sponge matrix. Each of these substrates consists of a very tions.112 The luciferase reporter technique requires that animals different surface, ranging from hard and inflexible, a gel, and are anesthetized and restrained so that sufficient photons a sponge-matrix, respectively. Folkman and Moscona119 can be collected to construct a pseudo-image. Detection found that cell shape was tightly coupled to DNA synthesis of luciferase-labeled cells in vivo is very low resolution.1 and cell growth. Therefore, the flexibility of a substrate is Therefore, cellular or subcellular imaging in vivo is not important for cells to maintain their optimal shape. Human possible with luciferase. The dependence on circulating luci- osteosarcoma cells, stably expressing a fusion protein of a(v) ferin makes the signal from luciferase imaging unstable.112 integrin and GFP, grew as a simple monolayer without any The luciferin substrate has to reach every cancer cell to be structure formation on the surface of a plastic dish. When the useful and the clearance of the luciferin results in an unstable osteosarcoma cells were cultured within Matrigelt, the signal.113,114 This process must be carried out in an almost cancer cells formed colonies but no other structures. When light-free environment. The requirement for photon count- the cancer cells were seeded on Gelfoams, the cells formed ing using luciferase precludes real-time imaging. The one three-dimensional tissue-like structures. The behavior of possible advantage of luciferase-based imaging is that no 143B osteosarcoma cells on Gelfoams in culture is excitation light is necessary. However, far-red absorbing remarkably different from those of these cells in mono-

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Figure 13 Transgenic RFP nude mouse. All of the major organs and tissues are red under fluorescence excitation with blue light. (a) Whole-body image of transgenic RFP nude mouse. Image was taken with a Hamamatsu C5810 tree-chip CCD camera. (b) Brain. (c) Heart and lungs. (d) Liver. (e) Circulatory system. (f) Uterus and ovary. (g) Pancreas. (h) Kidney and adrenal gland. (i) Spleen. (b–i) Images were taken with the Indec Biosystems FluorVivo imaging system.117

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Figure 14 Image rendering. Phase contrast fluorescence micrographs and rotating images of 143B human osteosarcoma cells expressing av integrin fused to GFP on plastic plates (left panels); in MatrigelTM culture (middle panels); in Gelfoams culture (right panels); using the Olympus FV1000 confocal microscope. Z-stack in 1 mm steps. The depths of images are on plastic surface 20 mm; MatrigelTM 70 mm; Gelfoams 180 mm. The images are rotated around the z axes from 01 to 801 in five sections, using the FV-10 ASW software system.120 layer culture or in Matrigelt. Tissue-like structures were fically excite GFP whose peak is distinct from that of the observed only in Gelfoams culture. A flexible structural skin, tissues, and fluid of the animal. In addition, proper substrate such as Gelfoams provides a more in vivo-like band-pass emission filters should be used with a cutoff culture condition than monolayer culture or Matrigelt. of approximately 515 nm.6,112,121 Matrigelt does not result in actual three-dimensional culture (Figure 14).120 ACKNOWLEDGMENTS It is important to minimize autofluorescence of tissue and These studies were supported in part by the National Cancer Institute body fluids by using proper filters. Excitation filters should grants CA132971 and CA142669 and CA183280. have a narrow band as close to 490 nm as possible to speci- DEDICATION: This article is dedicated to the memory of AR Moossa, MD.

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Figure 14 Continued.

DISCLOSURE/CONFLICT OF INTEREST Robert M Hoffman is an unsalaried affiliate of AntiCancer Inc.

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